The invention relates to novel mimetics of the tetrasaccharides sialyl- Lewis-X (SLeX) and sialyl-Lewis-A (SLeA), with improved action as inhibitors of cell adhesion, to a process for the preparation of these compounds and to their use as pharmacologically active compounds and diagnostic agents.
The circulation of blood cells, for example leukocytes, neutrophils, granulocytes and monocytes is, on the molecular level, a highly complex multistage process of which only individual steps are known (Review: T. A. Springer, Cell 76, 301-314,1994).
Very recent research results have shown that the recirculation of the lymphocytes, which is crucial in immune monitoring, and the localization of neutrophils and monocytes at inflammatory foci respond to very similar molecular mechanisms. Thus, in acute and chronic inflammatory processes, the leukocytes adhere to endothelial cells and migrate into the inflammatory focus and into the secondary lymphatic organs.
The leukocyte adhesion process involves numerous specific signal molecules, for example interleukins, leucotrienes and tumor necrosis factor (TNF), receptors coupled to their G protein, and, in particular, tissue-specific cell adhesion molecules, which ensure precisely controlled recognition of the immune cells and endothelial cells. The most important adhesion molecules involved in this process are the receptors and include the selectins (E-, P- and L-selectins), integrins and the members of the immunoglobulin superfamily.
The adhesion of leukocytes to endothelial cells, which is mediated by selectin receptors in the initial phase of inflammatory processes, is a natural and necessary immune response to various inflammatory stimuli and to damage to the vascular tissue.
The excessive adhesion of leukocytes and their infiltration into the effected tissue affects the progression of many acute and chronic diseases. For example, rheumatism, reperfusion injuries such as myocardial ischemia/infarction (MI), acute pneumonia after operative surgery, traumatic shock and stroke, psoriasis, dermatitis, ARDS (adult respiratory distress syndrome) and the restenosis occurring after surgical intervention (for example angioplasty and by-pass operations). Preventing or reducing the adhesion process at a very early stage of the inflammation, therefore, is a highly attractive and generally applicable concept for the pharmacological control of inflammatory diseases.
It is presently generally recognized that the tetrasaccharides sialyl-Lewis-X (SLeX) and sialyl-Lewis-A (SLeA), which occur as substructures of glycosphingolipids and glycoproteins on cell membranes, are able to function as ligands for all three selectin receptors. The literature describes a range of glycoproteins, mucins and glycolipids suitable for use as endogenous ligands of the selectins. These include the mucosal vascular addressin MadCAM-1 (Berg et al., Nature 1993, 366, 695) and the sialomucin CD34 (Baumhuter et al., Science 1993, 262, 436) for L-selectin, an O-linked polylactosamine sialomucin PSGL-1 on human neutrophils P-selectin (Moore et al., J. Biol. Chem. 1994, 269,23318) and N-linked sialoglycoproteins of the type ESL-1 for E-selectin (Vestweber et al., Cell Biol. 1993,121, 449).
The natural ligand having the structure of SLeX has already been successfully used in animal experiments in P-selectin-dependent lung injuries (M. S. Mulligan et al., Nature 1993, 364, 149) and in myocardial reperfusion injuries (M. Buerke et al., J. Clin. Invest. 1994, 93,1140). In primary clinical trials in the case of acute pneumonia the compound is to be employed in a dose of 1-2 grams per day per patient (communication from Cytel Corp./ La Jolla (Calif.) in the 2nd Glycotechnology Meeting/CHI in La Jolla/USA on May 16-18, 1994).
However, the specificity of these and other potential ligands for selectins in vivo has not yet been elucidated. The tetrasaccharides SLeX and SLeA which occur on selectin ligands represent only a substructure of the substantially more complex endogenous ligand structures and, owing to their very similar affinity for selectins, they cannot be used alone to explain a specific receptor binding.
The search for the specificity of the tetrasaccharide's SLeX and SLeA for selection has led to their preparation as pharmaceuticals in a various administrative forms. Moreover, the complexity of the endogenous ligand, of which SleX and SLeA is a substructure, has resulted in the search for mimetics having modfied structures from the natural ligand.
Efforts to obtain more strongly binding antagonists by structural variation of the ligand are an area of intense interest. The aim of such work is to provide more active antagonists which would also potentially be suitable for use in vivo at a low dose.
While the fucose and neuraminic acid units have been reported as crucial for the structure-activity relationship, their modification did not bring any significantly improved inhibition values. (B. K. Brandley et al., Glycobiology 1993, 3, 633 and M. Yoshida et al., Glycoconjugate J. 1993, 10, 3). Only when the glucosamine unit was varied (replacement of GlcNAc by glucose and azido and amino groups in position 2 of GlcNAc) was it possible to achieve a significantly increased affinity for the E-selectin receptor. Supra. In the case of the P-selectin receptor, in contrast, no improved binding was obtained. Supra.
In general, all successes to date in improving the binding affinity of SLeX derivatives and SLeA derivatives have been limited to the E-selectin receptor, since only weak inhibition effects have been found with the P-selectin receptor at inhibitor concentrations of about 1 mM (R. M. Nelson et al., J. Clin. Invest. 1993, 91, 1157).
The prior art relative to the binding affinity of modified SLeX/A structures for selectins is reviewed in Pharmacochem. Libr. 1993, 20 (Trends in Drug Research), pages 33-40.
In addition to the low binding affinity of these compounds for the selectins, however, they all comprise at least one unstable glycosidic linkage, which drastically limits the oral availability of these active compounds. This instability also greatly restricts the synthesis of different derivatives, since the sensitive glycosidic linkage, which has a tendency to be cleaved, imposes great restrictions on the reaction conditions. A wide variety of approaches have been taken to the synthesis of mimetics in order to obtain an increase in the stability.
However, an increase in the stability has been achieved by linking the side chain to the C-4 carbon of fucose via a C--C bond (Floyd et al., Tetrahedron Asymmetry 1994, 5, 2061).
Unfortunately, the linkage to C-4 of fucose also causes the orientation of the side chain to deviate from that of the natural ligand, as indicated in FIG. 1. As a result, only a very small affinity was evident for binding to the selectins. ##STR3##
The use of carbacyclic carbohydrate analogs in which the linkage of the side chain is by a C--C bond to C-1 would result in a conformation similar to that of the natural ligand, but yet would be stable to degradation. Work is being carried out with great intensity on the preparation of carbacyclic carbohydrate analogs of monosaccharide units.
Among the various methods those worthy of emphasis are the reactions of activated monosaccharides with nitromethane (Gross P. H., Tetrahedron 1991, 47,6113), allylsilane (Y. Kishi et al., J. Am. Chem. Soc. 104, 4976-4978, 1982) and olefins (D. A. Levy et al., Tetrahedron Asymmetry 5, 2265-2268, 1994).
The use of a carbacyclic analog as a building block for selectin antagonists led to a mimetic with affinity for selecting. In this context it was possible by reacting a fucose unit with allylsilane to synthesize a specific C-glycosidic unit 1 with a orientation of the side chain (WO 95/04751). With a/.beta.=14/1, the selectivities in the allylation are very good, but the proposed reaction conditions would not be readily amenable to scale up. Likewise, chromatographic purification, while necessary to purify the starting material, cannot be used to resolve the a/.beta. mixture. ##STR4##
The terminal acid function of the side chain of compound 1 is the attachment site for glycopeptide analogs, for example compound 2. The derivatives have IC.sub.50 values in the region of up to 1 mM. However, the glycopeptide analogs are unstable to the degradation of the peptide chain by proteases, so that the oral availability of these compounds is also highly limited. A further disadvantage is that the .beta. compound cannot be separated off in this synthesis. This is problematic because the .beta. derivatives are inactive as a result from the side chain having the wrong orientation, as demonstrated by corresponding model calculations. ##STR5##
In an analogous manner, the C-glycosidic unit 1 has also been used to synthesize various other mimetics which are intended to imitate the active conformation of SLe.sup.X. The compounds tested, for example compound 3, however, had IC.sub.50 values which at 10-20 mM were higher by a factor of 10-20 than that of the natural ligand SLe.sup.X (Wong et al., J. Am. Chem. Soc. 1995,117, 5395). ##STR6##
By using a substituted allylsilane it was possible to prepare the C-glycoside 4. ##STR7##
The triterpenoid acid derivative mimetics (betulinic acid: WO 95/04526; glycyrrhetinic acid: WO 94/24145) indicated by compound 4a were prepared and have multiple therapeutic effect. These compounds were tested and shown to display inhibition of 5-lipoxygenase, antimetastatic action and P-selectin inhibition. In these tests, the IC.sub.50 found for binding to P-selectin was 0.75 mM. However, it should be noted here that the triterpenoid acid alone gives values of 0.125 mM in this test. ##STR8##
The preparation of the C-glycoside unit in 4 also requires a complex chromatographic purification in the course of which the .beta. derivative can be separated only with difficulty. Moreover, the stability of the C-glycoside is reduced by the reactive allyl chloride group.
In addition to the preparative problems already described (complex chromatographic purification, high number of synthetic steps owing to the protective-group strategy, and the resolution of a/.beta. mixture), the abovementioned C-glycoside mimetics have a binding affinity for the selectins which is too small for them to intervene effectively in the adhesion processes. The reason for this is two-fold. First, the stability of a fucose mimetic as a freestanding molecule means that it will be less reactive. Second, the orientation and conformative fixation of the side chain in the aforementioned derivatives is not optimized for selectin binding. The importance of the side chain conformation is emphasized by Wong et al., where slight changes in the structure of the side chain have marked effects on the binding strength (O--C exchange).
According to L. A. Lasky, negatively charged sialic acid (or a negatively charged sulfonic acid group) is important for binding to selectins. Since the crystal structure of E-selectin has been clarified, investigations concerning possible binding sites have already been carried out. For example, sialic acid can be bound at lysines K111 and K113 (J. Bajorath et al., Biochemistry 1994, 33, 1332) as well as at Arg 97, Lys111 and Lys113 (Structural Biology 1, 140 (1994)).